The activities of many regulatory proteins and RNAs can be modulated by small molecule ligands that move freely within and between living cells. The mobility of these small molecules, and their effects on important cellular targets, make them excellent candidates for pharmaceutical development. Indeed, the National Institute of Health has declared a need to find small molecule ligands for every protein encoded in the genome. The discovery of therapeutic small molecules and their targets is also the primary focus of most pharmaceutical companies. The Nuclear Receptor (“NR”) superfamily of transcription factors comprises a particularly attractive set of small molecule targets. Unlike most other transcription factors, NRs are normally switched on and off by small lipids or lipophilic molecules. Furthermore, NRs feature in practically every fundamental biological process, functioning as key control points in key signaling and metabolic pathways {Mangelsdorf, 1995; Chawla, 2001}. The pivotal roles played by these proteins, and their potential for functional manipulation by natural and synthetic ligands, make them ideal targets for medical study and drug intervention.
The human genome contains 48 members of the NR superfamily. These proteins share a common structural organization, including a central, zinc finger DNA-binding domain (“DBD”), and C-terminal to this, a structurally conserved ligand-binding domain (“LBD”) {Kumar, 1999}. In addition to forming the ligand-binding pocket, the LBD is also involved in homo- and/or hetero-dimerization and possesses ligand-regulated binding sites for transcriptional co-activators and co-repressors {Egea, 2000; Wagner, 1995; Renaud, 1995; Uppenberg, 1998; Bledsoe, 2002; Dhe-Paganon, 2002; Gampe, 2000}. Most LBDs characterized to date are composed of approximately 12 α-helices arranged in three layers to form a hydrophobic ligand-binding pocket in the centre. Examples of NR ligands include steroid hormones, thyroid hormones, bile acids, fatty acids, certain vitamins and prostaglandins {Francis, 2003; Bogan, 1998}. Ligand binding induces structural changes in the LBD, such that, in the case of activators, the position of helix 12 is altered resulting in the displacement of co-repressors, recruitment of coactivators and subsequent target gene transcription {Glass, 2000}. Ligands whose binding promotes the transcriptional activation of target genes, termed agonists, induce different structural changes in LBDs than antagonists. Antagonists tend to possess bulky chemical groups that cannot be properly accommodated in the binding pocket, preventing the proper placement of helix 12 for coactivator binding {Glass, 2000}.
NRs play major roles in most physiological processes. These include sex determination {McElreavey, 1999}, maturation {Beuschlein, 2002}, growth control {Zhao, 2001}, metabolism {Basu-Modak, 1999}, neuronal growth and differentiation {Zhou, 1999; Satoh, 2002}, neuroendocrine function {Auger, 2000; Tetel, 2000} electrolyte homeostasis {Turnamian, 1990}, immune responses, xenobiotic responses {Kliewer, 1999; Kliewer, 2002; Willson, 2002}, circadian rhythm and aging {Pardee, 2004}. When NRs malfunction, major diseases ensue, some of which are listed below. By understanding NR functions and the ligands that control them, there is the potential to control the many diseases associated with inappropriate NR activity. There are already many very successful examples of this. One of the more familiar is the control of Estrogen Receptor (ER) activity in breast tumours by the synthetic antagonists tamoxifen and raloxifene {Tonetti, 1999; Osborne, 2000}. RXR- and RAR-directed retinoid analogs have proven successful in the treatment of acute promyelocytic leukemias {Degos, 1995} and glaucoma {Kim, 1990; Stoilov, 2001}. A great deal of attention has also been focused on the development of ligands for LXRs, PPARs and HNF4, which play major roles in hyperlipidaemia, atherosclerosis, diabetes and obesity {Kersten, 2000; Repa, 2002; Willson, 2001; Way, 2001; Wakino, 2002; Bogan, 2000}. Until recently, each of these NRs were orphans whose ligand(s) were unknown. Some of the most recent orphan NRs to gather attention are FXR, PXR and CAR, which all play major roles in xenobiotic responses {Stoilov, 2001; Willson, 2002; Xie, 2000; Kliewer, 1999; Kliewer, 2002; Kliewer, 2002}. Modulating the activities of these receptors can decrease occurrences of drug resistance and drug-drug incompatibility, which are major problems in drug treatment plans. Other major diseases known to be caused by inappropriate NR activity include Parkinson's disease {Satoh, 2002; Lee, 2002; Rawal, 2002}, cardiac myopathies {Zhu, 2003; Huss, 2002} and asthma {Bolt, 2001; Serhan, 2001}.
The potential of future NR-directed pharmaceuticals to control normal and abnormal biological processes is reflected by the percentage of top-selling drugs present on the market (>10%). This large presence is despite the fact that relatively few NRs have been successfully targeted. The large subfamily of orphan NRs yet to be targeted has the potential to define critical new biological processes and physiological pathways. Hence, they also represent an untapped resource for drug discovery and disease treatment. Even with the NRs that have known ligands, more potent, selective, stage- and tissue-specific agonists/antagonists need to be identified and developed. Further fine-tuning of drug specificities is also required to alleviate the cross-reactivity, cross talk and unwanted side effects of existing ligands. For example, tamoxifen, which is used to inactivate the Estrogen receptor (ER) in breast tumours, also blocks normal and necessary functions of the receptor in other tissues. Tamoxifen also cross-reacts with other NR. It is likely that new ER agonists and antagonists can be developed that act stage- and tissue-specifically and far more selectively.
The first NR ligands identified were hormones such as the insect metamorphosis-inducing steroid ecdysone and the female-specific steroid estrogen. The powerful effects of these hormones allowed for the large-scale and complex purification schemes necessary for their ultimate identification. However, the complexity of these purification procedures, the lack of suitable assays and the unsuitable biochemical nature of most ligands make conventional purification methods tedious, unsuitable or impossible for the remaining NR ligands.
Methods currently used to identify NR ligands involve in vitro based or cultured cell based screens. Most in vitro screening methods depend on ligand-mediated enhancement of coactivator peptide binding. One example of this is the ALPHAScreen, which makes use of time-resolved fluorescence resonance energy transfer (FRET) {Glickman, 2002}. The major drawback of all in vitro approaches, however, is that conditions and cofactors required for LBD stability and ligand and/or coactivator binding are likely to be missing. This means that many compounds with potential activity may be passed over. Also, compounds that do prove to be active in the test tube may prove unsuitable in vivo. For example, they may be unable to penetrate cells or tissues, they may be rapidly modified or degraded, or they prove to be toxic due to numerous off-target effects.
In the case of cultured cells, the most widely used screening methods involve ligand-stimulated reporter gene activation {Dias, 1998; Grover, 2003}. FRET has also been used to detect ligand-mediated LBD-cofactor interactions {Llopis, 2000; Day, 1998; Weatherman, 2002}. A yeast-based approach, that detects ligand mediated refolding of LBDs that are fused to yellow fluorescent protein (YFP), has also recently been developed {Muddana, 2003}. As with in vitro approaches, however, each of these cell-based assays is limited by the presence or absence of appropriate and inappropriate cofactors and conditions restricted to the particular cell type chosen for the assay. For example, alternative cofactors bound by a given NR in one cell type are often absent in others. In addition, the binding, delivery and function of some ligands only works in certain cell types. Also, as with the in vitro based assays, these compounds may prove to be unstable or toxic in a whole-animal system. Indeed, the majority of compound hits fail when subsequently tested in live model organisms. Thus in vitro and cultured cell based results, while high in throughput, tend not to be predictive of in vivo utility.
Further evidence that ligands with tissue-specific efficacy exist and are important is the observation that different ligands for tissue-specific receptors promote distinct changes in LBD conformations. These alternative conformations are likely to have different outcomes in different cell types due to the diversity of cofactors and cofactor modifications capable of interacting with these alternative conformations. Thus, a full understanding of in vivo NR function requires elucidation of the ligands that are active in each tissue of the living animal. Ultimately, this requires the testing of compounds in the entire organism during all stages of development, as well as adulthood and senescence.